U.S. patent number 6,825,047 [Application Number 09/627,580] was granted by the patent office on 2004-11-30 for device and method for multiple analyte detection.
This patent grant is currently assigned to Applera Corporation. Invention is credited to Michael Albin, Reid B. Kowallis, Robert P. Ragusa, Yefim Raysberg, Emily S. Winn-Deen, Timothy W. Woudenberg.
United States Patent |
6,825,047 |
Woudenberg , et al. |
November 30, 2004 |
**Please see images for:
( Certificate of Correction ) ** |
Device and method for multiple analyte detection
Abstract
The invention is directed to a method and device for
simultaneously testing a sample for the presence, absence, and/or
amounts of one or more a plurality of selected analytes. The
invention includes, in one aspect, a device for detecting or
quantitating a plurality of different analytes in a liquid sample.
The device includes a substrate which defines a sample-distribution
network having (i) a sample inlet, (ii) one or more detection
chambers, and (iii) channel means providing a dead-end fluid
connection between each of the chambers and the inlet. Each chamber
may include an analyte-specific reagent effective to react with a
selected analyte that may be present in the sample, and detection
means for detecting the signal. Also disclosed are methods
utilizing the device.
Inventors: |
Woudenberg; Timothy W. (Moss
Beach, CA), Albin; Michael (Antioch, CA), Kowallis; Reid
B. (Burlingame, CA), Raysberg; Yefim (Fremont, CA),
Ragusa; Robert P. (Los Altos, CA), Winn-Deen; Emily S.
(Potomac, MD) |
Assignee: |
Applera Corporation (Foster
City, CA)
|
Family
ID: |
33458425 |
Appl.
No.: |
09/627,580 |
Filed: |
July 28, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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012045 |
Jan 22, 1998 |
6124138 |
|
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831983 |
Apr 2, 1997 |
6126899 |
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Current U.S.
Class: |
436/518; 422/50;
422/504; 435/6.11; 436/164; 436/536; 436/546; 536/23.1 |
Current CPC
Class: |
B01L
3/50273 (20130101); B01L 3/502738 (20130101); C12Q
1/08 (20130101); G01N 35/08 (20130101); G01N
35/10 (20130101); B01L 3/5027 (20130101); B01L
3/5025 (20130101); Y10S 436/807 (20130101); B01L
7/52 (20130101); B01L 2200/0642 (20130101); B01L
2300/0816 (20130101); B01L 2300/0864 (20130101); B01L
2300/0887 (20130101); B01L 2400/049 (20130101); G01N
2035/00148 (20130101); G01N 2035/00356 (20130101); G01N
2035/1032 (20130101); G01N 2035/1034 (20130101) |
Current International
Class: |
B01L
3/00 (20060101); C12Q 1/06 (20060101); C12Q
1/08 (20060101); G01N 35/00 (20060101); G01N
033/543 (); G01N 021/00 (); G01N 033/536 (); C12Q
001/68 () |
Field of
Search: |
;436/518,164,536,546
;422/50,55,57,100,68.1 ;435/6,11 ;536/23.1 ;204/153.1,601
;250/458,458.1 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO93/22053 |
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WO93/22054 |
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WO |
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WO93/22055 |
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WO |
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WO93/22058 |
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WO |
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WO93/22421 |
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WO |
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WO94/11489 |
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WO |
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WO95/06508 |
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WO |
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WO95/06870 |
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WO |
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WO95/21382 |
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WO |
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WO96/03206 |
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Feb 1996 |
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WO |
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WO96/04547 |
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Feb 1996 |
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WO |
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Other References
Baileys and Scott, "Diagnotic Microbiology" 8th Edition, pp.
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Primary Examiner: Swartz; Rodney P
Assistant Examiner: Shahnan-Shah; Khatol
Attorney, Agent or Firm: Kilyk & Bowersox, P.L.L.C.
Parent Case Text
This application is a continuation of Ser. No. 09/012,045 filed
Jan. 22, 1998, now U.S. Pat. No. 6,124,138, which is a division of
Ser. No. 08/831,983 filed Apr. 2, 1997, now U.S. Pat. No.
6,126,899, which claims the benefit of priority of U.S. Provisional
Application Ser. No. 60/014,712 filed Apr. 3, 1996, all of which
are incorporated herein by reference.
Claims
What is claimed is:
1. A device for detecting or quantitating one or more of a
plurality of different polynucleotide sequences in a liquid sample,
said device comprising a substrate defining a sample-distribution
network having (i) a sample inlet, (ii) two or more detection
chambers, and (iii) channel means providing a dead-end fluid
connection between each of said chambers and said inlet, wherein at
least two of said detection chambers each contain a different,
sequence-specific polynucleotide binding polymer for detecting or
quantitating different polynucleotide sequences that may be present
in such sample, to produce a detectable signal, wherein said
substrate comprises two or more laminated layers, whereby
evacuation of said network, followed by application of such sample
to said inlet, is effective to draw sample by vacuum into each of
said chambers.
2. The device of claim 1, wherein said channel means comprises a
single channel to which said detection chambers are connected by
said fluid connections.
3. The device of claim 1, wherein said channel means comprises a
first channel to which a first group of detection chambers are
connected by such dead-end fluid connections, and a second channel
to which a second group of detection chambers are connected by such
dead-end fluid connections.
4. The device of claim 1, wherein said channel means comprises an
individual channel for each detection chamber, for providing a
dead-end fluid connection between said inlet and each detection
chamber.
5. The device of claim 1 comprising at least one laminated layer
comprising copper.
6. The device of claim 1 comprising at least one laminated layer
comprising aluminum.
7. The device of claim 1 comprising at least one laminated layer
comprising silicon.
8. The device of claim 1, wherein said detection means comprises an
optically transparent window associated with each detection
chamber, through which such signal can be optically detected.
9. The device of claim 1, wherein the at least one binding polymer
includes first and second oligonucleotide primers having sequences
effective to hybridize to opposite end regions of complementary
strands of a selected polynucleotide sequence, for amplifying the
sequence by primer-initiated polymerase chain reaction.
10. The device of claim 9, wherein the at least one binding polymer
further comprises a fluorescer-quencher oligonucleotide capable of
hybridizing to the selected polynucleotide sequence in a region
downstream of one of the primers, for producing a detectable
fluorescent signal when the selected sequence is present in the
sample.
11. The device of claim 1, wherein the at least one binding polymer
comprises first and second oligonucleotides effective to bind to
adjacent, contiguous regions of a selected polynucleotide
sequence.
12. The device of claim 11, wherein the at least one binding
polymer comprises a second pair of oligonucleotides which are
effective to bind to adjacent, contiguous regions complementary to
the regions bound by the first pair of oligonucleotides, for
amplification of the regions by ligase chain reaction.
13. The device of claim 1, wherein at least one of the detection
chambers additionally comprises an intercalating compound which
produces an optically detectable signal upon intercalating a
double-stranded polynucleotide.
14. The device of claim 1, wherein said substrate further comprises
temperature regulating means for controlling the temperature of
each detection chamber.
15. The device of claim 1, wherein said substrate defines at least
two such sample-distribution networks.
16. The device of claim 1, wherein the interior of said network is
under vacuum.
17. The device of claim 1, wherein at least one of the binding
polymers contains a fluorescent dye.
18. The device of claim 1, wherein at least one binding polymer
contains a fluorescent dye moiety which produces a detectable
signal upon hybridization of the binding polymer to a target
polynucleotide sequence.
19. The device of claim 1, wherein at least one binding polymer
comprises first and second oligonucleotides effective to bind to
adjacent regions of a selected polynucleotide sequence which are
separated from each other by one or more intervening bases.
20. The device of claim 1, wherein at least one of the two or more
laminated layers includes a high thermal conductivity layer.
21. The device of claim 20, wherein the high thermal conductivity
layer comprises at least one material selected from copper,
aluminum, and silicon.
22. The device of claim 20, further comprising a temperature
regulating device adapted to heat or cool the two or more detection
chambers by operatively contacting the high thermal conductivity
layer.
23. A device for detecting or quantitating one or more of a
plurality of different polynucleotide sequences in a liquid sample,
said device comprising a substrate defining a sample-distribution
network having (i) a sample inlet, (ii) two or more detection
chambers, and (iii) channel means providing a dead-end fluid
connection between each of said chambers and said inlet, wherein at
least two of said detection chambers each contain a different
sequence-specific polynucleotide binding polymer for detecting or
quantitating different polynucleotide sequences that may be present
in such sample, to produce a detectable signal, whereby a vacuum of
said network, followed by application of such sample to said inlet,
is effected to draw sample by vacuum into each of said chambers,
wherein the substrate includes a high thermal conductivity
surface.
24. The device of claim 23, wherein the high thermal conductivity
surface comprises at least one material selected from copper,
aluminum, and silicon.
25. The device of claim 24, further comprising a temperature
regulating device adapted to heat or cool the two or more detection
chambers by operatively contacting the high thermal conductivity
surface.
Description
FIELD OF THE INVENTION
The present invention relates to devices and methods for detecting
or quantifying one or more selected analytes in a sample.
REFERENCES Ausubel, F. M., et al., Current Protocols in Molecular
Biology, John Wiley & Sons, Inc., Media, PA. Bergot, J. B., et
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Buchardt, O., et al., PCT Pub. No. WO 92/20703 (1992). Froehler, et
al., Nucl. Acids Res. 16:4831 (1988). Fung, S., et al., EPO Pub.
No. 233,053 A2 (1987). Higuchi, R., et al., Bio/Technology 10:413
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BACKGROUND OF THE INVENTION
Biochemical testing is becoming an increasingly important tool for
detecting and monitoring diseases. While tests have long been known
for obtaining basic medical information such as blood type and
transplant compatibility, for example, advances in understanding
the biochemistry underlying many diseases have vastly expanded the
number of tests which can be performed. Thus, many tests have
become available for various analytical purposes, such as detecting
pathogens, diagnosing and monitoring disease, detecting and
monitoring changes in health, and monitoring drug therapy.
An important obstacle which has limited exploitation of many
biochemical tests has been cost. Simultaneous testing of multiple
samples for a single analyte has provided some savings. However,
simultaneous assays for a large number of analytes within a single
sample have been less practical because of the need for extended
sample manipulation, multiple test devices, multiple analytical
instruments, and other drawbacks.
Ideally, a method for analyzing an individual sample using a single
test device should provide diagnostic information for a large
number of potential analytes while requiring a small amount of
sample. The device should be small in size while providing
high-sensitivity detection for the analytes of interest. The method
should also require minimal sample manipulation. Preferably, the
device will include pre-dispensed reagents for specific detection
of the analytes.
SUMMARY OF THE INVENTION
The present invention is directed generally to a method and device
for simultaneously testing a sample for the presence, absence
and/or amount of one or more selected analytes.
The invention includes, in one aspect, a device for detecting or
quantitating one or more of a plurality of different analytes in a
liquid sample. The device includes a substrate which defines a
sample-distribution network having (i) a sample inlet, (ii) one or
more detection chambers, and (iii) channel means providing a
dead-end fluid connection between each of the chambers and the
inlet. Preferably, each chamber includes an analyte-specific
reagent effective to react with a selected analyte that may be
present in the sample, and detection means for detecting the
signal.
In one embodiment, the detection means for each chamber includes an
optically transparent window through which the signal can be
detected optically. In another embodiment, the detection means
includes a non-optical sensor for detecting the signal.
The channel means of the device may be configured in numerous ways.
For example, in one embodiment, the channel means includes a single
channel to which the detection chambers are connected by dead-end
fluid connections. In a second embodiment, the channel means
includes at least two different channels, each connected to a
different group of detection chambers. In yet another embodiment,
the channel means includes an individual channel for each detection
chamber.
The device may include a vacuum port for placing the detection
chambers under vacuum prior to the addition of sample. In one
embodiment, the vacuum port is connected to the channel means at a
site between, and in fluid communication with, the sample inlet and
the detection chambers. In another embodiment, the vacuum port is
connected to the channel means at a site downstream of the
detection chambers. In this configuration, the vacuum port is
additionally useful for removing liquid from the channel means
after the detection chambers have been filled, to help isolate the
detection chambers from one another and further reduce
cross-contamination.
The vacuum port may be incorporated in a multi-port valve (e.g., a
3-way valve) that permits the network and associated detection
chambers to be exposed alternately to a vacuum source, the sample
inlet, and a vent or selected gas source.
Alternatively, the device of the invention is prepared and sealed
under vacuum when manufactured, so that a vacuum port is
unnecessary.
According to an important feature of the invention, the device is
capable of maintaining a vacuum within the sample-distribution
network (low internal gas pressure, relative to the external,
ambient pressure outside the device) for a time sufficient to allow
a sample to be drawn into the network and distributed to the
detection chambers by vacuum action. For this purpose, the
sample-distribution network may include a vacuum reservoir in fluid
communication with, and downstream of, the detection chambers, for
preventing the build-up of back-pressure in the network while the
detection chambers are successively filled.
In one embodiment, the vacuum reservoir includes a non-flowthrough
cavity connected downstream of the last-filled detection chamber,
for accumulating residual gas displaced from the inlet and channel
means. In another embodiment, the reservoir comprises the terminal
end of the channel means connected to a vacuum source, allowing
residual gas displaced by the sample to be removed continuously
until sample loading is complete.
The analyte-specific reagents in the detection chambers may be
adapted to detect a wide variety of analyte classes, including
polynucleotides, polypeptides, polysaccharides, and small molecule
analytes, for example. In one embodiment, the analytes are
selected-sequence polynucleotides, and the analyte-specific
reagents include sequence-selective reagents for detecting the
polynucleotides. The polynucleotide analytes are detected by any
suitable method, such as polymerase chain reaction, ligase chain
reaction, oligonucleotide ligation assay, or hybridization
assay.
In one particular embodiment, for polynucleotide detection, the
analyte-specific reagents include an oligonucleotide primer pair
suitable for amplifying, by polymerase chain reaction, a target
polynucleotide region in the selected analyte which is flanked by
sequences complementary to the primer pair. The presence of target
polynucleotide, as indicated by successful amplification, is
detected by any suitable means.
In another embodiment, the analyte-specific reagents in each
detection chamber include an antibody specific for a selected
analyte-antigen. In a related embodiment, when the analyte is an
antibody, the analyte-specific detection reagents include an
antigen for reacting with a selected analyte antibody which may be
present in the sample.
In yet another embodiment, the device includes means for regulating
the temperatures of the detection chambers, preferably providing
temperature control between 0.degree. C. and 100.degree. C., for
promoting the reaction of the sample with the detection reagents.
In one preferred embodiment the temperature regulating means
includes a conductive heating element for each detection chamber,
for rapidly heating the contents of the chamber to a selected
temperature. The temperature control means is preferably adapted to
regulate the temperatures of the detection chambers, for heating
and cooling the chambers in accordance with a selected assay
protocol.
The device may be manufactured and packaged so that the
sample-distribution network (e.g., sample inlet, detection
chambers, and channel means) is provided under vacuum, ready for
immediate use by the user. Alternatively, the sample-distribution
network is provided under atmospheric pressure, so that the
evacuation step is carried out by the end-user prior to sample
loading.
The invention also includes a substrate containing a plurality of
sample-distribution networks as described above, for testing a
single sample or a plurality of samples for selected analytes.
In another aspect, the invention includes a method of making a
device such as described above.
In another aspect, the invention includes a method for detecting or
quantitating a plurality of analytes in a liquid sample. In the
method, there is provided a device of the type described above,
wherein the interior of the network is placed under vacuum. A
liquid sample is then applied to the inlet, and the sample is
allowed to be drawn into the sample-distribution network by vacuum
action, delivering sample to the detection chambers. The delivered
sample is allowed to react with the analyte-specific reagent in
each detection chamber under conditions effective to produce a
detectable signal when the selected analyte is present in the
sample. The reaction chambers are inspected or analyzed to
determine the presence and/or amount of the selected analytes in
the sample.
The device of the invention may also be provided as part of a kit
which additionally includes selected reagents, sample preparation
materials if appropriate, and instructions for using the
device.
These and other objects and features of the invention will be more
apparent from the following detailed description when read in light
with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A and 1B show a plan view (1A) and perspective view (1B) of
an exemplary assay device in accordance with the invention;
FIGS. 2A-2C illustrate several exemplary sample distribution
network configurations in accordance with the invention;
FIGS. 3A-3C illustrates a time sequence for the filling of the
detection chambers of a sample-distribution network with fluid
sample;
FIG. 4 illustrates a sample-distribution network containing three
sample delivery channels for delivering sample to three different
sets of detection chambers;
FIG. 5 illustrates a sample-distribution network having a separate
delivery channel for each detection chamber;
FIGS. 6A-6C illustrate selected features of another
sample-distribution network in accordance with the invention; the
device is shown in plan view (6A), perspective view (6B), with a
portion of the sample distribution network of the device shown in
FIG. 6C;
FIG. 7 shows an exploded view of a portion of a device in
accordance with the invention;
FIG. 8 shows an exploded view of a portion of another device in
accordance with the invention; and
FIG. 9 shows a perspective view of another device in accordance
with the invention.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
The following terms and phrases as used herein are intended to have
the meanings below.
"Dead-end fluid connection between a detection chamber and a
"sample inlet" refers to a fluid connection which provides the sole
fluid access to a detection chamber, such that fluid cannot enter
or exit the detection chamber by any other way than through the
dead-end fluid connection.
In particular, "dead-end fluid connection" refers to a channel
whose cross-section is sufficiently narrow to preclude
bi-directional fluid flow through the channel. That is, liquid
cannot flow through the channel in one direction while air or
another liquid is flowing through the channel in the opposite
direction.
As used herein, "microdevice" means a device in accordance with the
invention
II. Assay Device
In one aspect, the present invention provides a device which is
useful for testing one or more fluid samples for the presence,
absence, and/or amount of one or more selected analytes. The device
includes a substrate which defines a sample-distribution network
having (i) a sample inlet, (ii) one or more detection chambers
(preferably a plurality of detection chambers), and (iii) channel
means providing a dead-end fluid connection between each of the
chambers and the inlet. Each chamber includes an analyte-specific
reagent effective to react with a selected analyte that may be
present in such sample.
In one embodiment, the substrate also provides, for each chamber,
an optically transparent window through which analyte-specific
reaction products can be detected. In another embodiment, the
detection means for each chamber comprises a non-optical sensor for
signal detection.
The present invention provides a number of advantages in an assay
for multiple analytes in a sample, as will be discussed below. In
particular, the invention facilitates the transition from a macro
size sample to a micro-sized sample, wherein the device of the
invention provides one-step metering of reagents and sample in a
multi-analyte detection assay.
A. Network Configurations
FIGS. 1A and 1B show a plan view and perspective view,
respectively, of an exemplary assay device 30 in accordance with
the invention. Device 30 includes a substrate 32 which defines a
sample-distribution network 34. With reference to FIG. 1B, the
device also includes mount 36 containing a sample inlet 38 and
optionally, vacuum port means 40 which is located downstream of the
detection chambers.
Inlet 38 may be adapted to form a vacuum-tight seal with the end of
a syringe, for sample loading, or with a multi-port valve to
provide fluid communication with the sample and one or more liquid
or gaseous fluids. The inlet may further include a septum cap, if
desired, for maintaining the network under vacuum and allowing
introduction of sample by canula or needle.
Vacuum port 40 may be adapted for connection to a vacuum source,
such as a vacuum pump. The vacuum connection may include a valve
for closing off the sample-distribution network from the vacuum
source, or a multi-port valve for connection to a vacuum source and
one or more selected gas supplies.
Substrate 30 further provides indentations or holes 42, which may
be arranged asymmetrically as illustrated in FIG. 1A, to engage
corresponding pins or protrusions in a device-holder, not shown, to
immobilize and orient the device for analysis.
As noted in the Summary of the Invention, the sample-distribution
network of the invention may utilize any of a number of different
channel configurations, or channel means, for delivering sample to
the individual detection chambers. With reference to FIG. 2A,
distribution network 34a includes sample inlet 38a, vacuum port
means 40a, a plurality of detection chambers 44a, and channel means
comprising a single channel 46a to which the detection chambers are
each connected by dead-end fluid connections 48a. The detection
chambers are distributed on either side of channel 44a, with the
fluid-connections branching off in pairs from opposite sides of the
channel. FIG. 2B shows a portion of an alternative network 34b
having an inlet 38b and detection chambers 44B, wherein fluid
connections 48b branch off from channel 46b in a staggered
manner.
The detection chambers in the device of the invention may be
arranged to form a repeating 2 dimensional array which facilitates
indexing and identification of the various chambers, as well as
allowing rapid measurement of an optical signal produced by each
chamber upon reaction with the sample, if optical detection is
used.
FIGS. 2A-2B, for example, show networks in which the detection
chambers are arranged in rows and columns along perpendicular axes,
allowing the chambers to be identified by X and Y indices if
desired. This type of array (a perpendicular array) also
facilitates successive interrogations of the chambers in a
chamber-by-chamber analysis mode. However, other arrangements may
be used, such as a staggered or a close-packed hexagonal array.
FIG. 2C, for example, shows part of a network 34c having inlet 38c
and an array of staggered detection chambers 44c. The detection
chambers are connected to a common delivery channel 46c by fluid
connections 48c.
The device may also include identifying symbols adjacent the
detection chamber to facilitate identification or confirmation of
the analytes being detected.
Preferably, the detection chambers of the device are each provided
with analyte-specific reagents which is are effective to react with
a selected analyte which may be present in the sample, as discussed
further below. Reaction of the sample with the analyte-specific
reagents results in production of a detectable signal which
indicates that the selected analyte is present.
According to an important feature of the invention, the sample is
delivered to the detection chambers by vacuum action. Prior to
loading with sample, the interior of the sample-distribution
network is placed under vacuum so that the residual gas pressure in
the network is substantially below atmospheric pressure (i.e.,
substantially less than 760 mm Hg). One advantage of this feature
of the invention is that a pump for pushing fluid through the
network is not required. Instead, the device exploits ambient
atmospheric pressure to push the sample through the sample inlet
and into the sample-distribution network. This allows the sample to
be delivered quickly and efficiently to the detection chambers.
FIGS. 3A--3A illustrate the filling process for a
sample-distribution network 34 in accordance with FIG. 2A. The
network includes sample inlet 38, detection chambers 44, and sample
delivery channel 46 which is connected to the various detection
chambers by dead-end fluid connections 48. The network further
includes a vacuum reservoir 40 at the terminus of the delivery
channel. A plurality of the detection chambers 44 contain dried
detection reagents for detecting a different selected analyte in
each chamber, with one or more chambers optionally being reserved
as controls.
FIG. 3A shows the device before sample loading is initiated. The
network is evacuated to establish an internal pressure within the
network that is substantially below atmospheric pressure (e.g., 1
to 40 mm Hg). The interior of the network should also be
substantially liquid-free to minimize vapor pressure problems. FIG.
3B shows the network after sample fluid 50 has entered the network
through inlet 38 (FIG. 3B). As the sample moves through channel 46,
the sample sequentially fills each of the detection chambers (FIG.
3B) until all of the chambers have been filled (FIG. 3C). With
continued reference to FIG. 3C, once the detection chambers have
all been filled, sample fluid may continue to flow through channel
46 into vacuum reservoir 40 until the reservoir becomes full or the
flow is otherwise terminated (e.g., by closing a valve associated
with the vacuum reservoir).
According to one advantage of the invention, continued sample flow
through the channel means does not substantially disturb the
contents of the detection chambers that have already been filled,
because further flow into or out of each filled detection chamber
is restricted by the dead-end fluid connections, such as
connections 48. Cross-contamination between different detection
chambers is therefore reduced, so that erroneous signals due to
cross-contamination can be avoided. A further advantage of the
invention is that the sample can be mixed with the analyte-specific
detection reagents and detected all in the same chamber, without
requiring movement of the sample from each chamber to another site.
Moreover, since the sample and detection reagents can remain in the
chamber for signal detection, the detection reagents need not be
immobilized on or adhered to the inner surfaces of the detection
chambers.
The components of the sample-distribution network are designed to
ensure that an adequate volume of sample will be delivered to the
detection chambers to allow accurate analyte detection and/or
quantitation. In general, the percent-volume of a detection chamber
that must be occupied by the sample will vary according to the
requirements of the reagents and the detection system used.
Typically, the volume-percent will be greater than 75%, preferably
greater than 90%, and more preferably greater than 95%. In assay
formats in which the detection chambers are heated, particularly to
temperatures of between about 60.degree. C. and about 95.degree.
C., the volume-percent filling of the chambers is preferably
greater than 95%, and more preferably is at least 99%.
The degree to which the detection chambers are filled with sample
will generally depend upon (1) the initial ratio of the external
(atmospheric) pressure to the initial pressure within the network,
(2) the individual and total volumes defined by the detection
chambers, (3) the volume defined by the channel means, and (4) the
nature of the network downstream of the last detection chamber.
For example, in the case of a detection chamber which is nearest
the sample inlet, and which will be filled first, the percentage
occupancy (volume-percent) of sample fluid in the chamber after
sample loading (V.sub.s, %, will be related to the external
atmospheric pressure (P.sub.ext) and the initial internal pressure
within the network before sample loading (P.sub.int) by the
expression:
Thus, if the initial pressure within the network (P.sub.int) is 10
mm Hg, and the external pressure (P.sub.ext) is 760 mm Hg, about
99% of the first detection chamber will be filled with sample fluid
(V.sub.s, %.about.99%), with the remaining volume (.about.1.3%)
being filled by residual gas (e.g., air) displaced by the sample.
(This calculation assumes that, by the time the sample reaches the
chamber, the internal network pressure has not increased
appreciably due to displacement of gas upstream of the chamber.)
Similarly, if P.sub.ext is 760 mm Hg and P.sub.int is only 40 mm
Hg, the volume-percent of the chamber that becomes occupied with
sample will still be very high (about 95%).
It will be appreciated that as the sample fluid reaches and fills
successive detection chambers, the residual gas displaced from the
channel means will gradually accumulate in the remaining network
volume, so that the internal pressure will gradually increase. The
resultant increase in back-pressure can lead to a reduction in
V.sub.s, % for each successive chamber, with V.sub.s, % for the
last-filled detection chamber being significantly lower than the
V.sub.s, % for the first-filled chamber.
To help avoid this problem, the dimensions of the channel and
dead-end fluid connections are preferably selected to define a
total volume that is substantially less than the total volume
defined by the detection chambers. Preferably, the collective
volume of the channel means is less than 20% of the total
collective volume of the detection chambers, and more preferably
less than 5%. Similarly, the volume of each dead-end fluid
connection should be substantially less than the volume of the
associated detection chamber. Preferably, the volume of each
dead-end connection is less than 20%, preferably less than 10%, and
more preferably less than 5% of the volume of the associated
detection chamber.
The problem of back-pressure can be further diminished by including
a vacuum reservoir downstream of the last detection chamber to be
filled. In one embodiment, the vacuum reservoir is a
non-flowthrough cavity in which gas displaced by the sample fluid
can collect. The volume of the reservoir will vary according to the
configuration and needs of the particular device. For example, the
volume of the reservoir can be selected to be equal in volume to
one or more detection chambers volumes, or alternatively, is one-
to five-fold as great as the total collective volume of the channel
means.
In another embodiment, the vacuum reservoir is connected to a
vacuum source, so that residual gas can be removed continuously
until sample loading into the detection chambers is complete, as
discussed further below.
FIG. 4 shows another sample-distribution network in accordance with
the invention, wherein the channel means of the network includes at
least two different sample delivery channels, each connected to a
different group of detection chambers. FIG. 4 shows a
sample-distribution network 60 having a sample inlet 62, three
different groups of detection chambers 64a, 64b, and 64c, and
channel means 66 which include corresponding channels 66a, 66b, and
66c associated with the three chamber groups. The chambers are
connected to channels 64a-64c via dead-end fluid connections
68a-68c, which provide uni-directional flow of the sample into the
detection chambers.
One advantage of using multiple delivery channels is that the time
needed to fill the detection chambers with the sample can be
significantly reduced relative to the time needed to fill the same
number of detection chambers using a single delivery channel. For
example, the loading time for a network in accordance with FIG. 4
will be about one-third of that needed to fill an identical number
of detection chambers via the single channel format illustrated in
FIG. 2A, all other factors being equal. More generally, for a given
number of detection chambers, the filling time will vary inversely
with the number of delivery channels used.
The sample-distribution network in FIG. 4 further includes separate
vacuum reservoirs 70a-70c which are connected to the termini of
sample delivery channels 64a-64c, downstream of the detection
chambers. The vacuum chambers are dimensioned to help maintain a
low internal gas pressure during sample loading.
In another embodiment, the channel means includes an individual
channel for each detection chamber, as illustrated in FIG. 5.
Network 80 includes an inlet 82, detection chambers 84, and
associated with each detection chamber, a dead-end fluid connection
86, which may also be referred to as channel means, for delivering
sample to each chamber. Each dead-end fluid connection is
dimensioned to define a volume that is substantially less than the
volume of the associated detection chamber, to ensure that each
detection chamber is sufficiently filled with sample. This
embodiment provides rapid filling of the detection chambers with
minimal cross-contamination.
The device of the invention may also include a vacuum port
communicating with the sample-distribution network, for applying a
vacuum to the network before or during sample loading. In one
embodiment, the vacuum port is connected to the channel means at a
site between, and in fluid communication with, the sample inlet and
the detection chambers. An illustration of this can be found in
FIG. 9. The vacuum port thus provides a convenient way to reduce
the internal pressure within the network to a selected residual
pressure prior to sample loading. In particular, when the sample is
introduced into the network using a syringe barrel connected to the
sample inlet, the vacuum port can be used to remove air from the
space between the syringe and the inlet, before the sample is
admitted into the network.
In another embodiment, the vacuum port is connected to the channel
means at a site downstream of the sample inlet and detection
chambers (e.g., FIG. 6A). In this configuration, the vacuum port
may additionally be used to remove liquid from the channel means
after the detection chambers have been filled, to help isolate the
detection chambers from one another and further reduce
cross-contamination. In this configuration, the vacuum port
constitutes a part of the vacuum reservoir described above, where
the reservoir includes a vacuum source linked to the terminal end
of a sample delivery channel. The vacuum port may be kept open to
the network during sample loading, to continuously remove residual
gas from the network until all of the detection chambers have been
filled.
The vacuum port may include a multi-port valve (e.g., 3-way valve)
that permits the network and associated detection chambers to be
exposed alternately to a vacuum source, the sample inlet, and a
vent or gas source. Such a valve may be used to alternately expose
the network to vacuum and a selected gas source, to replace
residual air with the selected gas. Such gas replacement in the
network may be useful to remove molecular oxygen (O.sub.2) or other
atmospheric gases which might otherwise interfere with the
performance of the detection reagents.
Argon and nitrogen are inert gases which may be suitable for most
situations. Another gas which may be used is carbon dioxide
(CO.sub.2), which is highly soluble in water due to its ability to
form carbonate and bicarbonate ions. When the sample fluid is an
aqueous solution, bubbles of carbon dioxide which may form in the
network during sample loading may be eliminated via dissolution in
the sample fluid. The degree of sample filling in the detection
chambers is therefore enhanced. Of course, carbon dioxide should
not be used if it interferes with the detection reagents.
A multi-port valve such as noted above can also be used to supply a
gas which is required for detection of the selected analytes. For
example, it may be desirable to provide molecular oxygen or ozone
where the detection reagents involve an oxidation reaction. Other
gases, such as hydrocarbons (ethylene, methane) or nitrogenous
gases, may also be introduced as appropriate.
B. Device Fabrication
The device of the invention is designed to allow testing of a
sample for a large number of different analytes by optical
analysis, using a device that is compact and inexpensive to
prepare. Generally, the device will be no larger in cross-section
than the cross-section of a standard credit card (.ltoreq.5
cm.times.10 cm), and will have a thickness (depth) of no greater
than 2 cm. More preferably, the device occupies a volume of no
greater than about 5.times.5.times.1 cm, excluding attachments for
the sample inlet and any vacuum port. More preferably, the device
has dimensions of no greater than about 3 cm.times.2 cm.times.0.3
cm. Devices smaller than this are also contemplated, bearing in
mind that the device should provide adequate sensitivity and be
easy for the end-user to handle.
The detection chambers in the device are generally designed to be
as small as possible, in order to achieve high density of detection
chambers. The sizes and dimensions of the chambers will depend on a
number of considerations. When signal detection is by optical
means, the overhead cross-section of each chamber must be large
enough to allow reliable measurement of the signal produced when
the selected analyte is present in the sample. Also, the depths of
the chambers can be tailored for the particular optical method
used. For fluorescence detection, thin chambers may be desirable,
to minimize quenching effects. For absorbance or chemiluminescence
detection, on the other hand, a thicker chamber may be appropriate,
to increase the detection signal.
It will be appreciated that while the figures in the attached
drawings show chambers having square-shaped overhead
cross-sections, other geometries, such as circles or ovals, may
also be used. Similarly, the channels in the network may be
straight or curved, as necessary, with cross-sections that are
shallow, deep, square, rectangular, concave, or V-shaped, or any
other appropriate configuration.
Typically, the detection chambers will be dimensioned to hold from
0.001 .mu.L to 10 .mu.L of sample per chamber, and, more preferably
between 0.01 .mu.L and 2 .mu.L. Conveniently, the volume of each
detection chamber is between about 0.1 .mu.L and 1 .mu.L, to allow
visual confirmation that the chambers have been filled. For
example, a chamber having a volume of 0.2 .mu.L may have dimensions
of 1 mm.times.1 mm.times.0.2 mm, where the last dimension is the
chamber's depth.
The sample delivery channels are dimensioned to facilitate rapid
delivery of sample to the detection chambers, while occupying as
little volume as possible. Typical cross-sectional dimensions for
the channels will range from 5 .mu.m to about 250 .mu.m for both
the width and depth. Ideally, the path lengths between chambers
will be as short as possible to minimize the total channel volume.
For this purpose (to minimize volume), the network is preferably
substantially planar, i.e., the channel means and detection
chambers in the device intersect a common plane.
The substrate that defines the sample-distribution network of the
invention may be formed from any solid material that is suitable
for conducting analyte detection. Materials which may be used will
include various plastic polymers and copolymers, such as
polypropylenes, polystyrenes, polyimides, and polycarbonates.
Inorganic materials such as glass and silicon are also useful.
Silicon is especially advantageous in view of its high thermal
conductivity, which facilitates rapid heating and cooling of the
device if necessary. The substrate may be formed from a single
material or from a plurality of materials.
The sample-distribution network is formed by any suitable method
known in the art. For plastic materials, injection molding will
generally be suitable to form detection chambers and connecting
channels having a desired pattern. For silicon, standard etching
techniques from the semiconductor industry may be used, as
described in Sze (1988), for example.
Typically, the device substrate is prepared from two or more
laminated layers, as will be discussed below with reference to
FIGS. 6A-6C to 8. For optical detection, the device will include
one or more layers which provide an optically transparent window
for each detection chamber, through which the analyte-specific
signal is detected. For this purpose, silica-based glasses, quartz,
polycarbonate, or an optically transparent plastic layer may be
used, for example. Selection of the particular window material
depends in part on the optical properties of the material. For
example, in a fluorescence-based assay, the material should have
low fluorescence emission at the wavelength(s) being measured. The
window material should also exhibit minimal light absorption for
the signal wavelengths of interest.
Other layers in the device may be formed using the same or
different materials. Preferably, the layer or layers defining the
detection chambers are formed predominantly from a material that
has high heat conductivity, such as silicon or a heat-conducting
metal. The silicon surfaces which contact the sample are preferably
coated with an oxidation layer or other suitable coating, to render
the surface more inert. Similarly, where a heat-conducting metal is
used in the substrate, the metal can be coated with an inert
material, such as a plastic polymer, to prevent corrosion of the
metal and to separate the metal surface from contact with the
sample. The suitability of a particular surface should be verified
for the selected assay.
For optical detection, the opacity or transparency of the substrate
material defining the detection chambers will generally have an
effect on the permissible detector geometries used for signal
detection. For the following discussion, references to the "upper
wall" of a detection chamber refer to the chamber surface or wall
through which the optical signal is detected, and references to the
"lower wall" of a chamber refers to the chamber surface or wall
that is opposite the upper wall.
When the substrate material defining the lower wall and sides of
the detection chambers is optically opaque, and detection is by
absorption or fluorescence, the detection chambers will usually be
illuminated and optically scanned through the same surface (i.e.,
the top surfaces of the chambers which are optically transparent).
Thus, for fluorescence detection, the opaque substrate material
preferably exhibits low reflectance properties so that reflection
of the illuminating light back toward the detector is minimized.
Conversely, a high reflectance will be desirable for detection
based on light absorption.
When the substrate material defining the upper surface and sides of
the detection chambers is optically clear, and detection involves
fluorescence measurement, the chambers can be illuminated with
excitation light through the sides of the chambers (in the plane
defined collectively by the detection chambers in the device), or
more typically, diagonally from above (e.g., at a 45 degree angle),
and emitted light is collected from above the chambers (i.e.,
through the upper walls, in a direction perpendicular to the plane
defined by the detection chambers). Preferably, the substrate
material exhibits low dispersion of the illuminating light in order
to limit Rayleigh scattering.
When the entirety of the substrate material is optically clear, or
at least the upper and lower walls of the chambers are optically
clear, the chambers can be illuminated through either wall (upper
or lower), and the emitted or transmitted light is measured through
either wall as appropriate. Illumination of the chambers from other
directions will also be possible as already discussed above.
With chemiluminescence detection, where light of a distinctive
wavelength is typically generated without illumination of the
sample by an outside light source, the absorptive and reflective
properties of the substrate will be less important, provided that
the substrate provides at least one optically transparent window
for detecting the signal.
FIGS. 6A-6C illustrate a specific embodiment of a device in
accordance with the invention. With reference to FIGS. 6A and 6B,
device 100 includes a sample inlet 102, sample-distribution network
104, and vacuum port 106 which is connected to the terminus of
network 104. Network 104 includes a perpendicular array of
detection chambers 108 (7 rows.times.8 columns) linked to sample
delivery channel 110 via dead-end fluid connections 112. The device
further includes vertical panel 114 adjacent sample inlet 102, for
attaching an identifying label to the device and as an attachment
allowing the user to hold the device.
As can be seen from FIG. 6B, the detection chambers are packed
closely together to increase the number of analytes which can be
tested in the device. Fluid connections 112 are provided in an
L-shaped configuration (FIG. 6C) to impede fluid flow out of the
chambers after sample loading, and to help isolate the contents of
the chambers from each other. Although the horizontal rows of
detection chambers in FIGS. 6A and 6B are shown as being separated
from each other by variable vertical spacing (to enhance the
clarity of the figures), it will be appreciated that the chambers
can be separated by equal distances in both the vertical and
horizontal directions, to facilitate analysis of the chambers.
FIGS. 7 and 8 illustrate two exemplary approaches for forming a
testing device in accordance with FIGS. 6A-6B. FIG. 7 shows two
substrate layers 140 and 142 which can be brought together to form
sample-distribution network 104 (FIG. 6A). The network is defined
primarily by substrate layer 140, which contains indentations
defining a sample inlet 102 (not shown), a plurality of detection
chambers 108, sample delivery channel 110, and dead-end fluid
connections 112. Contact of substrate layer 142 with the opposing
face of layer 140 completes the formation of network 104.
FIG. 8 shows substrate layers 150 and 152 for forming a network by
another approach. Substrate layer 150 contains indentations
defining a plurality of detection chambers 108. Substrate layer
152, on the other hand, contains indentations defining sample
delivery channel 110 and dead-end fluid connections 112. Network
104 can then be formed by contacting the opposing faces of the two
substrate layers as in FIG. 7.
Since the device is designed to provide a vacuum-tight environment
within the sample-distribution network for sample loading, and also
to provide detection chambers having carefully defined reaction
volumes, it is desirable to ensure that the network and associate
detection chambers do not leak. Accordingly, lamination of
substrate layers to one another should be accomplished so as to
ensure that all chambers and channels are well sealed.
In general, the substrate layers can be sealably bonded in a number
of ways. Conventionally, a suitable bonding substance, such as a
glue or epoxy-type resin, is applied to one or both opposing
surfaces that will be bonded together. The bonding substance may be
applied to the entirety of either surface, so that the bonding
substance (after curing) will come into contact with the detection
chambers and the distribution network. In this case, the bonding
substance is selected to be compatible with the sample and
detection reagents used in the assay. Alternatively, the bonding
substance may be applied around the distribution network and
detection chambers so that contact with the sample will be minimal
or avoided entirely. The bonding substance may also be provided as
part of an adhesive-backed tape or membrane which is then brought
into contact with the opposing surface. In yet another approach,
the sealable bonding is accomplished using an adhesive gasket layer
which is placed between the two substrate layers. In any of these
approaches, bonding may be accomplished by any suitable method,
including pressure-sealing, ultrasonic welding, and heat curing,
for example.
The device of the invention may be adapted to allow rapid heating
and cooling of the detection chambers to facilitate reaction of the
sample with the analyte-detection reagents. In one embodiment, the
device is heated or cooled using an external
temperature-controller. The temperature-controller is adapted to
heat/cool one or more surfaces of the device, or may be adapted to
selectively heat the detection chambers themselves.
To facilitate heating or cooling with this embodiment, the
substrate material of the test device is preferably formed of a
material which has high thermal conductivity, such as copper,
aluminum, or silicon. Alternatively, a substrate layer such as
layer 140 in FIG. 7 may be formed from a material having moderate
or low thermal conductivity, while substrate layer 142 (FIG. 7) is
provided as a thin layer, such that the temperature of the
detection chambers can be conveniently controlled by heating or
cooling the device through layer 142, regardless of the thermal
conductivity of the material of layer 142. In one preferred
embodiment, layer 142 is provided in the form of an adhesive
copper-backed tape.
In an alternative embodiment, means for modulating the temperature
of the detection chambers is provided in the substrate of the
device itself. For example, with reference to FIG. 7, substrate
layer 142 may include resistive traces which contact regions
adjacent the reaction chambers, whereby passage of electical
current through the traces is effective to heat or cool the
chambers. This approach is particularly suitable for silicon-based
substrates, and can provide superior temperature control.
Further illustration of the invention is provided by the device
shown in FIG. 9. Device 160 includes a network-defining substrate
layer 161 and a flat substrate layer 180 for bonding with and
sealing layer 161.
Layer 161 includes sample inlet 162 and indentations defining (i) a
sample-distribution network 164 and (ii) vacuum reservoir 166
connected to the terminus of network 164. Network 164 includes a 2
dimensional perpendicular array of detection chambers 168
(7.times.7) linked to sample delivery channel 170 via dead-end
fluid connections 172. The device further includes vertical panel
174 adjacent sample inlet 162, as in FIGS. 6A-6B. Formation of
network 164 is completed by contacting the entirety of the upper
surface of device 160 with the opposing face of a layer 180, which
is preferably provided in the form of a membrane or thin layer.
Device 160 in FIG. 9 is distinguished from device 100 in FIG. 6A in
that device 160 includes a vacuum reservoir 166, instead of a
vacuum port, at the terminus of delivery channel 170. In addition,
sample inlet 162 in device 160 is conveniently adapted to operate
in conjunction with inlet fitting 190, so that evacuation of the
network and sample loading can be effected from a single site with
respect to the network.
Sample inlet 162 includes a hollow inlet cylinder 176 having an
open proximal end 177, which connects to network 172, and an open,
distal end 178. Cylinder 176 further includes an opening 179
located near the terminus of the distal end.
Inlet fitting 190 includes an inlet cap structure 200 and a port
structure 210 appended thereto. Cap structure 200 defines a hollow
cylinder 202 having an open, proximal end 204 and a closed, distal
end 206. The inner diameter of cylinder 202 is dimensioned to form
a vacuum-tight seal when placed over inlet 162. Port structure 210
defines a vacuum port 212 and a sample port 214. Ports 212 and 214
communicate with cylinder 202 via openings 216 and 218,
respectively, which are formed in the side of cylinder 202. Fitting
190 additionally includes guide structure 220 for receiving the
adjacent edge of panel 174, to orient and guide fitting 190 when
fitting 190 is fitted over and slided along inlet 162.
Exemplary dimensions of a device which has been prepared in
accordance with FIG. 9 are the following: detection chambers 168,
1.2 mm.times.1.2 mm.times.0.75 mm; delivery channel 170, 0.25
mm.times.0.25 mm (width.times.depth); dead-end fluid connection
172, 0.25 mm.times.0.25 mm (width.times.depth); external
dimensions: 22 cm.times.15 cm.times.1 mm (dimensions of
network-defining portion, excluding inlet 162 and panel 174).
Preferably, the detection chambers in the microdevice of the
invention have volumes less than 10 .mu.L, less than 2 .mu.L, and
most preferably less than or equal to 1 .mu.L.
Device 160 may be prepared under ordinary atmospheric-conditions by
bonding a polymeric, adhesive-backed substrate layer 180 to the
corresponding surface of layer 161, to form a sealed network. Inlet
fitting 190 is fitted onto cylinder 176 of inlet 162, such that
openings 179 and 216 are aligned with each other. Vacuum port 212
is connected to a vacuum line, and the interior of the network is
evacuated for a selected time. The network may be alternately
flushed with a selected gas, such as carbon dioxide, and vacuum, as
discussed above. During evacuation, sample port 214 is loaded with,
or placed in fluid communication with, the fluid sample.
Preferably, the sample port is filled so that there is no air
between the sample in the sample port and opening 218. Once the
network has been evacuated (usually complete within a few seconds),
fitting 190 is lowered further towards layer 161 until opening 179
is aligned with sample opening 218, bringing the interior of the
network in fluid communication with the sample. The sample fills
the chambers rapidly, typically in less than half a second. The
detection chambers are filled to a volume-percent of greater than
95%. Excess sample and residual gas collects in reservoir 166.
Once the chambers have filled with sample, fitting 190 is lowered
further toward layer 161 in order to seal inlet 162, thereby
sealing the interior of the network from the outside atmosphere.
The sample is allowed to react with the detection reagents in the
detection chambers, during or after which the optical signals
produced in the chambers are detected.
C. Detection Reagents
The detection chamber(s) of the device may be pre-loaded with
detection reagents which are specific for the selected analytes of
interest. The detection reagents are designed to produce an
optically detectable signal via any of the optical methods noted in
Section II below.
It will be appreciated that although the reagents in each detection
chamber must contain substances specific for the analyte(s) to be
detected in the particular chamber, other reagents necessary to
produce the optical signal for detection may be added to the sample
prior to loading, or may be placed at locations elsewhere in the
network for mixing with the sample. Whether particular assay
components are included in the detection chambers or elsewhere will
depend on the nature of the particular assay, and on whether a
given component is stable to drying. In general, it is preferred
that as many of the detection reagents as possible are pre-loaded
in the detection chambers during manufacture of the device, in
order to enhance assay uniformity and minimize the assay steps
conducted by the end-user.
The analyte to be detected may be any substance whose presence,
absence, or amount is desireable to be determined. The detection
means can include any reagent or combination of reagents suitable
to detect or measure the analyte(s) of interest. It will be
appreciated that more than one analyte can be tested for in a
single detection chamber, if desired.
In one embodiment, the analytes are selected-sequence
polynucleotides, such as DNA or RNA, and the analyte-specific
reagents include sequence-selective reagents for detecting the
polynucleotides. The sequence-selective reagents include at least
one binding polymer which is effective to selectively bind to a
target polynucleotide having a defined sequence.
The binding polymer can be a conventional polynucleotide, such as
DNA or RNA, or any suitable analog thereof which has the requisite
sequence selectivity. For example, binding polymers which are
analogs of polynucleotides, such as deoxynucleotides with
thiophosphodiester linkages, and which are capable of base-specific
binding to single-stranded or double-stranded target
polynucleotides may be used. Polynucleotide analogs containing
uncharged, but stereoisomeric methylphosphonate linkages between
the deoxyribonucleoside subunits have been reported (Miller, 1979,
1980, 1990, Murakami, Blake, 1985a, 1985b). A variety of analogous
uncharged phosphoramidate-linked oligonucleotide analogs have also
been reported (Froehler). Also, deoxyribonucleoside analogs having
achiral and uncharged intersubunit linkages (Stirchak) and
uncharged morpholino-based polymers having achiral intersubunit
linkages have been reported (U.S. Pat. No. 5,034,506). Binding
polymers known generally as peptide nucleic acids may also be used
(Buchardt, 1992). The binding polymers may be designed for sequence
specific binding to a single-stranded target molecule through
Watson-Crick base pairing, or sequence-specific binding to a
double-stranded target polynucleotide through Hoogstein binding
sites in the major groove of duplex nucleic acid (Kornberg). A
variety of other suitable polynucleotide analogs are also
known.
The binding polymers for detecting polynucleotides are typically
10-30 nucleotides in length, with the exact length depending on the
requirements of the assay, although longer or shorter lengths are
also contemplated.
In one embodiment, the analyte-specific reagents include an
oligonucleotide primer pair suitable for amplifying, by polymerase
chain reaction, a target polynucleotide region of the selected
analyte which is flanked by 3'-sequences complementary to the
primer pair. In practicing this embodiment, the primer pair is
reacted with the target polynucleotide under hybridization
conditions which favor annealing of the primers to complementary
regions of opposite strands in the target. The reaction mixture is
then thermal cycled through several, and typically about 20-40,
rounds of primer extension, denaturation, and primer/target
sequence annealing, according to well-known polymerase chain
reaction (PCR) methods (Mullis, Saiki).
Typically, both primers for each primer pair are pre-loaded in each
of the respective detection chambers, along with the standard
nucleotide triphosphates, or analogs thereof, for primer extension
(e.g., ATP, CTP, GTP, and TTP), and any other appropriate reagents,
such as MgCl.sub.2 or MnCl.sub.2. A thermally stable DNA
polymerase, such as Taq, Vent, or the like, may also be pre-loaded
in the chambers, or may be mixed with the sample prior to sample
loading. Other reagents may be included in the detection chambers
or elsewhere as appropriate. Alternatively, the detection chambers
may be loaded with one primer from each primer pair, and the other
primer (e.g., a primer common to all of detection chambers) may be
provided in the sample or elsewhere.
If the target polynucleotides are single-stranded, such as
single-stranded DNA or RNA, the sample is preferably pre-treated
with a DNA- or RNA-polymerase prior to sample loading, to form
double-stranded polynucleotides for subsequent amplification.
The presence and/or amount of target polynucleotide in a detection
chamber, as indicated by successful amplification, is detected by
any suitable means. For example, amplified sequences may be
detected in double-stranded form by including an intercalating or
crosslinking dye, such as ethidium bromide, acridine orange, or an
oxazole derivative, for example, which exhibits a fluorescence
increase or decrease upon binding to double-stranded nucleic acids
(Sambrook, 1989; Ausubel; Higuchi, 1992, 1993; Ishiguro, 1995).
The level of amplification can also be measured by fluorescence
detection using a fluorescently labeled oligonucleotide, such as
disclosed in Lee et al. (1993) and Livak et al. (1995). In this
embodiment, the detection reagents include a sequence-selective
primer pair as in the more general PCR method above, and in
addition, a sequence-selective oligonucleotide (FQ-oligo)
containing a fluorescer-quencher pair. The primers in the primer
pair are complementary to 3regions in opposing strands of the
target analyte segment which flank the region which is to be
amplified. The FQ-oligo is selected to be capable of hybridizing
selectively to the analyte segment in a region downstream of one of
the primers and is located within the region to be amplified.
The fluorescer-quencher pair includes a fluorescer dye and a
quencher dye which are spaced from each other on the
oligonucleotide so that the quencher dye is able to significantly
quench light emitted by the fluorescer S at a selected wavelength,
while the quencher and fluorescer are both bound to the
oligonucleotide. The FQ-oligo preferably includes a 3'-phosphate or
other blocking group to prevent terminal extension of the 3end of
the oligo.
The fluorescer and quencher dyes may be selected from any dye
combination having the proper overlap of emission (for the
fluorescer) and absorptive (for the quencher) wavelengths while
also permitting enzymatic cleavage of the FQ-oligo by the
polymerase when the oligo is hybridized to the target. Suitable
dyes, such as rhodamine and fluorscein derivatives, and methods of
attaching them, are well known and are described, for example, in
Menchen et al. (1993, 1994), Bergot et al. (1991), and Fung et al.
(1987).
The fluorescer and quencher dyes are spaced close enough together
to ensure adequate quenching of the fluorescer, while also being
far enough apart to ensure that the polymerase is able to cleave
the FQ-oligo at a site between the fluorescer and quencher.
Generally, spacing of about 5 to about 30 bases is suitable, as
generally described in Livak et al. (1995). Preferably, the
fluorescer in the FQ-oligo is covalently linked to a nucleotide
base which is 5' with respect to the quencher.
In practicing this approach, the primer pair and FQ-oligo are
reacted with a target polynucleotide (double-stranded for this
example) under conditions effective to allow sequence-selective
hybridization to the appropriate complementary regions in the
target. The primers are effective to initiate extension of the
primers via DNA polymerase activity. When the polymerase encounters
the FQ-probe downstream of the corresponding primer, the polymerase
cleaves the FQ-probe so that the fluorescer is no longer held in
proximity to the quencher. The fluorescence signal from the
released fluorescer therefore increases, indicating that the target
sequence is present.
One advantage of this embodiment is that only a small proportion of
the FQ-probe need be cleaved in order for a measurable signal to be
produced. In a further embodiment, the detection reagents may
include two or more FQ-oligos having distinguishable fluorescer
dyes attached, and which are complementary for different-sequence
regions which may be present in the amplified region, e.g., due to
heterozygosity (Lee, 1993).
In another embodiment, the detection reagents include first and
second oligonucleotides effective to bind selectively to adjacent,
contiguous regions of a target sequence in the selected analyte,
and which may be ligated covalently by a ligase enzyme or by
chemical means (Whiteley, 1989; Landegren, 1988) (oligonucleotide
ligation assay, OLA). In this approach, the two oligonucleotides
(oligos) are reacted with the target polynucleotide under
conditions effective to ensure specific hybridization of the
oligonucleotides to their target sequences. When the
oligonucleotides have base-paired with their target sequences, such
that confronting end subunits in the oligos are basepaired with
immediately contiguous bases in the target, the two oligos can be
joined by ligation, e.g., by treatment with ligase. After the
ligation step, the detection wells are heated to dissociate
unligated probes, and the presence of ligated, target-bound probe
is detected by reaction with an intercalating dye or by other
means.
The oligos for OLA may also be designed so as to bring together a
fluorescer-quencher pair, as discussed above, leading to a decrease
in a fluorescence signal when the analyte sequence is present.
In the above OLA ligation method, the concentration of a target
region from an analyte polynucleotide can be increased, if
necessary, by amplification with repeated hybridization and
ligation steps. Simple additive amplification can be achieved using
the analyte polynucleotide as a target and repeating denaturation,
annealing, and ligation steps until a desired concentration of the
ligated product is achieved.
Alternatively, the ligated product formed by hybridization and
ligation can be amplified by ligase chain reaction (LCR), according
to published methods (Winn-Deen). In this approach, two sets of
sequence-specific oligos are employed for each target region of a
double-stranded nucleic acid. One probe set includes first and
second oligonucleotides designed for sequence-specific binding to
adjacent, contiguous regions of a target sequence in a first strand
in the target. The second pair of oligonucleotides are effective to
bind (hybridize) to adjacent, contiguous regions of the target
sequence on the opposite strand in the target. With continued
cycles of denaturation, reannealing and ligation in the presence of
the two complementary oligo sets, the target sequence is amplified
exponentially, allowing small amounts of target to be detected
and/or amplified.
In a further modification, the oligos for OLA or LCR assay bind to
adjacent regions in a target polynucleotide which are separated by
one or more intervening bases, and ligation is effected by reaction
with (i) a DNA polymerase, to fill in the intervening single
stranded region with complementary nucleotides, and (ii) a ligase
enzyme to covalently link the resultant bound oligonucleotides
(Segev, 1992, 1994).
In another embodiment, the target sequences can be detected on the
basis of a hybridization-fluorescence assay (Lee et al., 1993). The
detection reagents include a sequence-selective binding polymer
(FQ-oligo) containing a fluorescer-quencher pair, as discussed
above, in which the fluorescence emission of the fluorescer dye is
substantially quenched by the quencher when the FQ-oligo is free in
solution (i.e., not hybridized to a complementary sequence).
Hybridization of the FQ-oligo to a complementary sequence in the
target to form a double-stranded complex is effective to perturb
(e.g., increase) the fluorescence signal of the fluorescer,
indicating that the target is present in the sample. In another
embodiment, the binding polymer contains only a fluorescer dye (but
not a quencher dye) whose fluorescence signal either decreases or
increases upon hybridization to the target, to produce a detectable
signal.
It will be appreciated that since the selected analytes in the
sample will usually be tested for under generally uniform
temperature and pressure conditions within the device, the
detection reagents in the various detection chambers should have
substantially the same reaction kinetics. This can generally be
accomplished using oligonucleotides and primers having similar or
identical melting curves, which can be determined by empirical or
experimental methods as are known in the art.
In another embodiment, the analyte is an antigen, and the
analyte-specific reagents in each detection chamber include an
antibody specific for a selected analyte-antigen. Detection may be
by fluorescence detection, agglutination, or other homogeneous
assay format. As used herein, "antibody" is intended to refer to a
monoclonal or polyclonal antibody, an Fc portion of an antibody, or
any other kind of binding partner having an equivalent
function.
For fluorescence detection, the antibody may be labeled with a
fluorescer compound such that specific binding of the antibody to
the analyte is effective to produce a detectable increase or
decrease in the compound's fluorescence, to produce a detectable
signal (non-competitive format). In an alternative embodiment
(competitive format), the detection means includes (i) an
unlabeled, analyte-specific antibody, and (ii) a fluorescer-labeled
ligand which is effective to compete with the analyte for
specifically binding to the antibody. Binding of the ligand to the
antibody is effective to increase or decrease the fluorescence
signal of the attached fluorescer. Accordingly, the measured signal
will depend on the amount of ligand that is displaced by analyte
from the sample. Exemplary fluorescence assay formats which may be
adapted for the present invention can be found in Ullman (1979,
1981) and Yoshida (1980), for example.
In a related embodiment, when the analyte is an antibody, the
analyte-specific detection reagents include an antigen for reacting
with a selected analyte antibody which may be present in the
sample. The reagents may be adapted for a competitive or
non-competitive type format, analogous to the formats discussed
above. Alternatively, the analyte-specific reagents include a mono-
or polyvalent antigen having one or more copies of an epitope which
is specifically bound by the antibody-analyte, to promote an
agglutination reaction which provides the detection signal.
In yet another embodiment, the selected analytes are enzymes, and
the detection reagents include enzymesubstrate molecules which are
designed to react with specific analyte enzymes in the sample,
based on the substrate specificities of the enzymes. Accordingly,
detection chambers in the device each contain a different substrate
or substrate combination, for which the analyte enzyme(s) may be
specific. This embodiment is useful for detecting or measuring one
or more enzymes which may be present in the sample, or for probing
the substrate specificity of a selected enzyme. Particularly
preferred detection reagents include chromogenic substrates such as
NAD/NADH, FAD/FADH, and various other reducing dyes, for example,
useful for assaying hydrogenases, oxidases, and enzymes that
generate products which can be assayed by hydrogenases and
oxidases. For esterase or hydrolase (e.g., glycosidase) detection,
chromogenic moieties such as nitrophenol may be used, for
example.
In another embodiment, the analytes are drug candidates, and the
detection reagents include a suitable drug target or an equivalent
thereof, to test for binding of the drug candidate to the target.
It will be appreciated that this concept can be generalized to
encompass screening for substances that interact with or bind to
one or more selected target substances. For example, the assay
device can be used to test for agonists or antagonists of a
selected receptor protein, such as the acetylcholine receptor. In a
further embodiment, the assay device can be used to screen for
substrates, activators, or inhibitors of one or more selected
enzymes. The assay may also be adapted to measure dose-response
curves for analytes binding to selected targets.
The sample or detection reagents may also include a carrier
protein, such as bovine serumalbumin (BSA) to reduce non-specific
binding of assay components to the walls of the detection
chambers.
The analyte-specific detection reagents are preferably dispensed
into the detection chambers robotically using a dispensing system
designed to deliver small volumes of liquid solutions (e.g., 0.1 to
1 .mu.L). The system is supplied with separate analyte-specific
detection reagents which are dispensed to preselected detection
chambers without cross-contamination.
A reagent loading device that has been prepared in accordance with
the invention includes a dispensing robot (Asymtek Automove 402)
coupled to a plurality of drop-on-demand ink-jet printing heads.
The robot includes an X,Y-axis work table (12 inch.times.12 inch)
having a lateral resolution of 0.001 inch, a lateral velocity of
0-20 inch/sec, a Z-axis resolution of 0.001 inch, and a Z-axis
velocity of 0-8 inch/sec. The robot optionally includes a tip
locator, offset camera, strobe drop camera, on-axis camera, and/or
gravimetric drop calibration. The printing heads are of a drop on
demand, piezo-electric type, having wetted surfaces usually
selected from glass, Teflon.RTM., and polypropylene. The minimum
drop volume is 25 nL, and the maximum flow is 1 .mu.L/min.
Reagent loading is preferably accomplished under
carefully-controlled sterile conditions using one or more dedicated
dispensing robots. After application, the reagents are allowed to
dry in the chambers until most or all of the solvent has
evaporated. Drying may be accelerated by baking or reduced pressure
as appropriate. The detection chambers are then sealed by bonding
the chamber containing substrate layer with an appropriate cover
layer, and the device is ready for use.
III. Signal Detection and Analysis
The signal produced by reaction of the analyte-specific reagents
with the sample is measured by any suitable detection means,
including optical and non-optical methods.
Where the signal is detected optically, detection may be
accomplished using any optical detector that is compatible with the
spectroscopic properties of the signal. The assay may involve an
increase in an optical signal or a decrease. The optical signal may
be based on any of a variety of optical principals, including
fluorescence, chemiluminescence, light absorbance, circular
dichroism, optical rotation, Raman scattering, radioactivity, and
light scattering. Preferably, the optical signal is based on
fluorescence, chemiluminescence, or light absorbance.
In general, the optical signal to be detected will involve
absorbance or emission of light having a wavelength between about
180 nm (ultraviolet) and about 50 .mu.m (far infrared). More
typically, the wavelength is between about 200 nm (ultraviolet) and
about 800 nm (near infrared). A variety of detection apparatus for
measuring light having such wavelengths are well known in the art,
and will typically involve the use of light filters,
photomultipliers, diode-based detectors, and/or charge-coupled
detectors (CCD), for example.
The optical signals produced in the individual detection chambers
may be measured sequentially by iteratively scanning the chambers
one at a time or in small groups, or may be measured simultaneously
using a detector which interrogates all of the detection chambers
continuously or at short time intervals. Preferably, the signals
are recorded with the aid of a computer capable of displaying
instantaneously (in real-time) the signal level in each of the
detection chambers, and also storing the time courses of the
signals for later analysis.
The optical signal in each chamber may be based on detection of
light having one or more selected wavelengths with defined
band-widths (e.g., 500 nm.+-.5 nm). Alternatively, the optical
signal may be based on the shape or profile of emitted or absorbed
light in a selected wavelength range. Preferably, the optical is
signal will involve measurement of light having at least two
distinctive wavelengths in order to include an internal control.
For example, a first wavelength is used to measure the analyte, and
a second wavelength is used to verify that the chamber is not empty
or to verify that a selected reagent or calibration standard is
present in the detection chamber. An aberration or absence of the
signal for the second wavelength is an indication that the chamber
may be empty, that the sample was improperly prepared, or that the
detection reagents are defective.
In studies conducted in support of the invention, a detection
assembly was prepared for fluorescence detection of target
polyrucleotides in a sample using a device in accordance with the
invention. The assembly includes a translation stage for
positioning the test device. The test device includes a 7.times.7
array of addressable detection chambers containing fluorescent
detection reagents. The detector in the assembly consists of a
tungsten bulb (or quartz halogen bulb, 75 W) illumination source, a
CCD camera, and appropriate focusing/collection optics. The
illumination source is positioned so as to illuminate the device
diagonally from above (e.g., at an inclination angle of 45 degrees
with respect to the illuminated surface). The optics include two
lenses separated by an emission filter. The first lens collimates
the incoming image for the emission filter, and the second lens
which re-images the filtered beam onto the CCD. The test device is
placed at the focal point of the first lens.
The CCD is a thermoelectrically cooled, instrumentation-grade
front-illuminated CCD (Princeton Instruments TEA/CCD-512TK/1). The
detection plate of the CCD has a 512.times.512 array of 27 .mu.m
square pixels which covers the entire overhead cross-section of the
test device. The camera head is controlled by a controller
(Princeton Instruments ST-135) which communicates with a computer
(Quadra 650, Apple Computers) for collecting and processing the
signal data. The system is capable of on-chip binning of the
pixels. For detection chambers having an overhead cross-section of
1 mm.times.1 mm, bins having a size of 2.times.2 pixels are
suitable. More generally, the bin size is selected on the basis of
the total processing time that will be required, the sizes and
number of detection chambers, sensitivity, and signal noise.
The computer in the assembly includes signal-processing software
for selecting an appropriate sub-region in each detection chamber
from which the signal is measured. Such sub-regions are selected
for uniformity of incoming light, to ensure that edge regions are
excluded. The signal image of the device is recorded and stored at
selected intervals, according to the requirements of the assay.
Preferably, the signal for each detection chamber is recorded as an
average signal per bin for the selected sub-region in each chamber,
since the size of the selected sub-region in each chamber will
usually differ from chamber to chamber.
The detector optics may further be adapted to include a filter
wheel for detecting fluorescence at 2 or more wavelengths.
As discussed above, the temperature of the detection chambers may
be controlled, if appropriate, by any of a number of suitable
methods. In the detection assembly that was prepared in accordance
with the invention, the heating means is external to the test
device (off-chip heating), and includes a temperature controller
(Marlow Industries model SE 5020) equipped with a peltier device
(ITI Ferro Tec model 6300) having a ramp rate of about
.+-.4.degree./sec in the range of 55.degree. C. to 95.degree. C.
For on-chip heating, where the device includes resistive tracings
(or a comparable equivalent) for heating the chambers, the assembly
can be modified to provide one or two zones of resistance heating
capable of establishing a maximum power dissipation of 25 W over a
200 mm.sup.2 area; this mode can provide a ramp rate of
.+-.10.degree./sec during transition from 55.degree. C. to
95.degree. C.
The above described structure (Section IIB) for detecting an
analyte-related signal in each chamber, including the optical
window associated with that chamber, is also referred to herein
collectively as detection means for detecting such signal.
Another type of detection means is a biosensor device capable of
detecting the reaction of an analyte with an analyte-specific
reagent in each chamber. Amperometric biosensors suitable for use
in the invention operate on a variety of principles. In one, the
analyte being measured is itself capable of interacting with an
analyte-specific reagent to generate an electrochemical species,
i.e., a species capable of function as an electron donor or
acceptor when in contact with an electrode. As an example, reaction
of the analyte cholesterol with the reagent S cholesterol oxidase
generates the electrochemical species H.sub.2 O.sub.2 which, in
contact with an electrode, produces a measurable current in a
circuit containing the electrode.
The analyte-specific reagent may be localized on a film separated
from the electrode surface by a permselective layer that is
selectively permeable to the electrochemical species (and other
small components in the sample). When sample fluid is added to the
biosensor, reaction of the analyte with the is corresponding
reagent produces an electrochemical species whose presence and
amount are quantitated by current measurement through the
electrode.
Alternatively, the analyte-specific reagent may be a receptor which
is specific for the analyte. Initially, the receptor sites are
filled with an analyte-enzyme conjugate. In the presence of
analyte, the conjugates are displaced from the receptor, and are
then free to migrate to positions close to the electrode, for
production of transient electrochemical species (such as H.sub.2
O.sub.2 in the presence of catalase) in the vicinity of the
electrode.
Another general type of biosensor employs a lipid bilayer membrane
as a gate for electrochemical species interposed between a
sample-fluid chamber and an electrode. The bilayer is provided with
ion-channel proteins which function as ion gates that can be opened
by analyte binding to the proteins. Thus, binding of analyte to the
channel proteins (which serve as the analyte-specific reagent)
leads to ion flow across the membrane and detectable signal at the
electrode.
Thin-film biosensors of the type mentioned above may be formed in a
microchip or small-substrate format by photolithographic methods,
such as described in U.S. Pat. Nos. 5,391,250, 5,212,050,
5,200,051, and 4,975,175. As applied to the present invention, the
chamber walls in the substrate may serve as the substrate for
deposition of the required electrode and film layers. In addition
to these layers, suitable conductive connectors connecting the
electrodes to electrical leads are also laid down.
In a typical device, each chamber contains a biosensor for a given
analyte. When sample is introduced into the device, the multiple
sample analytes are then separately measured in the chambers, with
the results being reported to a processing unit to which the device
is electrically connected.
IV. Assay Method
In another aspect, the invention includes a method for detecting or
quantitating a plurality of analytes in a liquid sample. In the
method, there is provided a device of the type described above,
wherein the interior of the network is placed under vacuum. A
liquid sample is then applied to the inlet, and the sample is
allowed to be drawn into the sample-distribution network by vacuum
action, delivering sample to the detection chambers. The delivered
sample is allowed to react with the analyte-specific reagent in
each detection chamber under conditions effective to produce a
detectable signal when the selected analyte is present in the
sample. The reaction chambers are inspected or analyzed to
determine the presence and/or amount of the selected analytes in
the sample.
The sample tested may be from any source which can be dissolved or
extracted into a liquid that is compatible with the device, and
which may potentially contain one or more of the analytes of
interest. For example, the sample may be a biological fluid such as
blood, serum, plasma, urine, sweat, tear fluid, semen, saliva,
cerebral spinal fluid, or a purified or modified derivative
thereof. The sample may also be obtained from a plant, animal
tissue, cellular lysate, cell culture, microbial sample, or soil
sample, for example. The sample may be purified or pre-treated if
necessary before testing, to remove substances that might otherwise
interfere with analyte detection. Typically, the sample fluid will
be an aqueous solution, particularly for polar analytes such as
polypeptides, polynucleotides, and salts, for example. The solution
may include surfactants or detergents to improve analytes
solubility. For non-polar and hydrophobic analytes, organic
solvents may be more suitable.
As discussed above, the device may be manufactured and sold in a
form wherein the sample-distribution network is under vacuum, so
that the device is ready to load by the end-user. Alternatively,
evacuation of the network is conducted by the user, through a
vacuum port or via the sample inlet itself.
Prior to sample loading, any gas in the network may be replaced
with another gas, according to the requirements of the assay. In a
preferred embodiment, the residual gas is replaced with carbon
dioxide, so that any gas bubbles that appear in the network after
sample loading are quickly dissolved by the sample fluid,
particularly if the sample is an aqueous solution.
It will be appreciated that when the device includes a vacuum port
downstream of the detection chambers, the sample delivery channels
in the device may be cleared of sample after the detection chambers
have been filled, to further isolate the detection chambers from
each other. The invention also contemplates filling the delivery
channels with an additional fluid, such as a mineral oil or a
viscous polymer solution containing agarose or other viscous
material (e.g., see Dubrow, U.S. Pat. No., 5,164,055, and Menchen
et al., U.S. Pat. No. 5,290,418), to segregate the chambers from
each other, or with a reagent-containing solution which facilitates
the assay.
In a particularly advantageous embodiment of the invention, a
large-volume syringe can be used to generate a vacuum inside the
sample distribution network of the device prior to loading. By
"large-volume" is meant that the volume of the syringe is greater
than the total internal volume of the device (i.e., of the sample
distribution network). Preferably, the volume of the syringe is at
least 20-fold greater than the interior volume of the device. With
reference to the device in FIG. 9, the inlettip of the syringe is
connected to vacuum port 212. When opening 216 is aligned with
opening 179, the syringe is used to draw air from the interior of
the device, thereby lowering the internal pressure. For example, if
a syringe with a volume of 50 mL is used, and the internal volume
of the device is 100 .mu.L, the pressure in the sample distribution
network can be reduced by a factor of 500 (.about.0.1 mL/50 mL).
Thus, an initial internal pressure of 760 torr can be reduced to
less than 2 torr. With reference to the device in FIG. 6A, the
syringe can be connected to fitting 106 or 102 using appropriate
connections, to withdraw air from the distribution network.
Accordingly, the present invention includes a kit comprising (i) a
device as described above and (ii) a syringe for drawing air from
the interior of the device. The invention also includes a method of
using the kit to detect one or more analytes in a sample, as
described above. It will be appreciated that using a syringe
greatly simplifies the step of creating a vacuum inside the device,
so that the device can be used quickly or immediately without
needing a mechanical vacuum pump.
V. Utility
The present invention can be used in a wide variety of
applications. The invention can be used for medical or veterinary
purposes, such as detecting pathogens, diagnosing or monitoring
disease, genetic screening, determining antibody or antigen titers,
detecting and monitoring changes in health, and monitoring drug
therapy. The invention is also useful in a wide variety of
forensic, environmental, and industrial applications, including
screening drug candidates for activity.
More generally, the present invention provides a convenient method
for simultaneous assay of multiple analytes in a sample. The
invention is highly flexible in its applications, being adaptable
to analysis of a wide variety of analytes and sample materials. By
providing pre-dispensed, analyte-specific reagents in separate
detection chambers, the invention eliminates the need for
complicated and time-consuming reagent preparation.
Practice of the invention is further simplified since the detection
chambers can be loaded via a single sample inlet. The use of
uniformly sized detection chambers renders the device
self-metering, in that a precise volume of sample is delivered to
each chamber. Thus, the precision, accuracy and reproducibility of
the assay are all enhanced, since the quantities and compositions
of the analyte-specific reagents, the quantity of sample in the
chambers, and the reaction conditions can be carefully controlled.
Moreover, very small volumes of sample are required since the
dimensions of the sample-distribution network in the device can be
very small.
The device may be formed from a wide variety of materials, allowing
the composition of the device to be adapted to the particular
reagents and conditions in the assay. Inasmuch as the device
requires no moving parts, and can be relatively small in size
(typically having dimension on the order of millimeters to
centimeters), manufacture of the device is simplified and costs are
reduced.
The features and advantages of the invention may be further
understood from the following example, which is not intended in any
way to limit the scope of the invention.
EXAMPLE
The following study was performed using a polycarbonate microdevice
substantially as shown in FIG. 9, to demonstrate detection of a
human .beta.-actin gene by PCR (polymerase chain reaction). The
assay components for PCR detection were obtained from PE Applied
Biosystems (Foster City, Calif., .beta.-actin kit, part
#N808-0230). The kit components included the following stock
solutions: .beta.-actin forward primer:
3 .mu.M primer in 10 mM Tris-HCl, pH 8.0 (at room temperature), 1
mM EDTA .beta.-actin reverse primer:
3 .mu.M primer in 10 mM Tris-ECl, pH 8.0 (at room temperature), 1
mM EDTA .beta.-actin probe:
2 .mu.M TAMRA-labeled probe in 10 mM Tris-HCl, pH 8.0 (at room
temperature), 1 mM EDTA DNA sample:
370 .mu.g/mL human genomic DNA in 10 mM Tris-HCl, pH 8.0 (at room
temperature), 1 mM EDTA (from Coriell Cell Repositories, Camden,
N.J.) dNTPs:
20 mM dNTP (1 tube each for dUTP, dATP, dCTP and dGTP) in
autoclaved deionized ultrafiltered water, titrated to pH 7.0 with
NaOH DNA polymerase:
"AMPLITAQ GOLD" DNA polymerase at 5 U/.uparw.L, from PE Applied
Biosystems, part # N808-0240 (PE Applied Biosystems "AMPLITAQ GOLD"
Product Brochure, 1996) "AMPERASE" UNG:
uracil-N-glycosylase at 1 U/.mu.L, from PE Applied Biosystems, part
#N808-0096 10.times. "TAQMAN" buffer A:
500 mM KCl, 100 mM Tris-HCl, 0.1 M EDTA, 600 nM Passive Reference 1
(ROX), pH 8.3 at room temperature, autoclaved MgCl.sub.2 :
20 mM MgCl.sub.2 in autoclaved deionized ultrafiltered water.
A description of the sequences of the forward primer, the reverse
primer, and the TAMRA-labeled probe can be found in PE Applied
Biosystems "TAQMAN" PCR Reagent Protocol (1996), which also
describes the general steps of the "TAQMAN" assay technique. The
forward and reverse primers were effective to produce a 297
basepair PCR product.
A flat substrate layer 180 and a substrate layer 161 were formed
from polycarbonate by standard injectionmolding methods (substrate
layer 161) or from sheet stock (layer 180). The volume of each
detection chamber was 1 .mu.L.
Detection chambers were loaded with different amounts of forward
primer, reverse primer, and fluorescent probe as follows. To a
polypropylene tube was added 0.5 mL each of .beta.-actin forward
primer solution, reverse primer solution, and fluorescent probe, to
give a final primer/probe stock solution volume of 1.5 mL. This
solution was then loaded into alternating detection chambers in
substrate layer 161 using a robotically controlled microsyringe.
Specifically, alternate chambers were loaded with either a
1.times., 5.times. or 10.times. amount of primer/probe solution,
with 1.times. (14 nL primer/probe stock solution) being equivalent
to a final concentration in a detection chamber of 15 nM of each
primer and 10 nM of fluorescent probe (after the dried chamber is
subsequently filled with sample), 5.times. (72 nL primer/probe
stock solution) being equivalent to a final concentration of 75 nM
of each primer and 50 nM of fluorescent probe, and 10.degree. (145
nL primer/probe stock solution) being equivalent to a final
concentration of 150 nM of each primer and 100 nM of fluorescent
probe. The amounts of primer and probe in the loaded chambers
corresponded to 1/20, 1/4, and 1/2 of the concentrations used under
standard reaction conditions, for the lx, 5.times., and 10.times.
chambers, respectively. The loaded chambers produced a
"checkerboard" pattern in substrate layer 161 where each loaded
chamber was separated by an intervening empty chamber.
After the loaded chambers were allowed to air-dry to dryness at
room temperature, the loaded substrate layer (161) was joined to a
flat substrate layer 180 by ultrasonic welding. Inlet fitting 190
was then placed over sample inlet 162, such that opening 179 was
aligned with vacuum port opening 216. The sample distribution
network 164 and detection chambers 168 were evacuated via vacuum
port 212, which was connected to a vacuum pump, to a final internal
pressure of approximately 1 to 10 torr.
The PCR reaction mixture (sample), without primers and probe, was
prepared from the above stock solutions to give the following final
concentrations in the sample:
10 mM Tris-HCl, pH 8.3
50 mM KCl
3.5 mM MgCl,
400 .mu.M dUTP
200 .mu.M each dATP, dCTP, and dGTP
0.01 U/.mu.L uracil-N-glycosylase
0.25 U/.mu.L "AMPLITAQ GOLD" DNA polymerase
0.74 ng/.mu.L human genomic DNA template
For loading of sample into the microdevice, a micropipette loaded
with the above sample solution was placed in sample port 214 so as
to minimize the deadvolume occupied by air at the tip of the
pipette. Inlet fitting 190 was then pressed down further to align
opening 179 with opening 218, so that the sample was drawn from
port 214 into the detection chambers by vacuum action. Filling of
the chambers was complete in less than a second.
The microdevice was then clamped to a peltier device (20
mm.times.20 mm) glued to an aluminum heat sink. Cycling was
controlled using a Marlow temperature controller (Marlow Industries
Inc., Dallas, Tex., Model No. SE 5020). A thermistor was attached
to the peltier device to provide temperature feedback (Marlow part
No. 217-2228-006). The microdevice was thermocycled as follows: 1)
precycle: 50.degree. C. for 2 minutes; 95.degree. C. for 10
minutes; 2) 40 cycles: 95.degree. C. for 15 seconds, 60.degree. C.
for 1 minute; 3) hold at 72.degree. C.
Signal detection was accomplished using a fluorescence detection
instrument consisting of a tungsten bulb for illumination and a CCD
camera and 4-color filter wheel for detection. Images of all
detection chambers (wells) were taken at the end of each
thermocycle (during the 60.degree. C. step) at several wavelengths
in order to monitor the increase of the reporter's fluorescence.
Interfering fluorescence fluctuations were normalized by dividing
the emission intensity of the reporter dye by the emission
intensity of the passive reference (ROX dye) for a given chamber.
The excitation wavelength was 488 nm. The reporter intensity was
measured at 518 nm, and the passive reference intensity was
measured at 602 nm.
Results. Positive fluorescent signals were detected in all chambers
that had been loaded with the .beta.-actin primers and fluorescent
probe at the 5.times. and 10 concentrations. Little or no signal
was detected for chambers loaded at the 1.times. concentration. No
detectable signal was detected above background for the chambers
which did not contain .beta.-actin primer and probe, indicating
that there was no cross-contamination between detection chambers
after 40 heat/cool cycles.
The highest final fluorescence signals were obtained in detection
chambers loaded with a 10.times. amount of primers and probe, with
detectable signals appearing after about 23 cycles. The
5.times.chambers also showed detectable signals after cycle 23, but
the final fluorescence signal was not as high as that for the
10.times. wells (due to lower probe concentration). Thus, the
.beta.-actin gene was readily detected using primer and probe
concentrations equal to 1/4 and 1/2 of those used under ordinary
conditions. The results also show that the preloaded primers and
probes were successfully dissolved in the sample after sample
loading.
Although the invention has been described by way of illustration
and example for purposes of clarity and understanding, it will be
appreciated that various modifications can be made without
departing from the invention. All references cited above are
incorporated herein by reference.
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